Tissue engineering of implants is a long and risky process with respect to maintaining sterility, which implies procurement of cells from the donor, transfer of the cells to a laboratory and manipulation of such cells to initiate expansion and/or differentiation. Following the expansion period cells are frequently removed from a temporary attachment substrate by i.e. trypsinization and thereafter transferred onto a scaffold and again cultured on this scaffold. This process therefore requires often not only days but weeks to be effective.
The rapid and correct manufacturing of complex 3D grafts is presently not known in the art. It is fundamental and indeed contradictory to current teaching, which focuses on cell technologies to expand and differentiate cells to trigger commitment in vitro and by seeding them thereafter onto scaffolds or growing them on these scaffolds directly. It is expected that those cells differentiate in vitro. This process often requires at least 1-2 weeks in average or even more.
A second line of teaching uses injecting methods for undifferentiated stem cells from bone marrow or blood or crude bone marrow as a form of cell therapy intra-operatively directly into a tissue including e.g. the heart muscle. For the repair of spinal injury cells were either cultured for expansion or from specific sources such as the nose or embryonic origin. The latter have the risk to go into transformation or tumor formation. The nose derived cells represent a rather infectious environment for harvesting and could not convince clinically as a generic solution. Bone marrow derived cells are under investigation.
A further alternative is de novo tissue regeneration. It was expected that the local environment will eventually help to differentiate these cells. Examining the receiving environment more closely it is found that the cells do not differentiate into e.g. heart muscle cells after injection into the heart. In these instances no formation of muscle cells was reported at all. Instead a rather positive effect from the secretory activity of the transplanted stem cells for support of recovery was postulated. Overall effects in such studies was a 4% improvement of cardiac function only. This means that this microenvironment hypothesis does not achieve the goal of tissue de novo formation but has an adjuvant role only.
In another study expanded MSC were injected after expansion in vitro into a acellularized valve scaffold. During in vitro culture cells underwent a selection process that achieved to select cells that have a prominent stem cell character (stronger replication) and that may lead to a reduced inflammation in vitro.
So far it is not clear what roles cytokines may play in this context. However it is well known from prior art that molecules in vitro can be used to control multipotency and to induce differentiation and commitment to a specific tissue.
Wound healing is closely linked to inflammatory responses. After surgical implantation of an artificial trachea the speed and quality of local healing, survival and integration is crucial for graft take and the long term success of the implant.
During inflammation cytokines are released such as IL-6, IL-1 and TNF that sustain the inflammatory response. Inflammation nevertheless can be a two-edged sword, if inflammation is not terminated in due course due to insufficient remodelling of the implant scaffold.
Scaffold remodeling in tissue engineering was conceived of as a rather unknown process and triggering mechanisms were either obscure or clinically not feasible. Conventionally cells would have been seeded onto the material of choice and integration into this material was a process that was attributed to ideally cell expansion time and migratory penetration.
Fundamentally, there is a positive side to inflammation being a perquisite for healing that needs to be taken into consideration for biological-implant engineering.
The prior art does not provide adequate teaching on the controlling either of the microenvironment after transplantation of the graft for sustainable remodeling, differentiation of undifferentiated cells after transplantation and of stem cells sensing the wound zones and does not teach adequately the differentiation of transplanted cells after expanded in vitro to achieve true scar free healing.
Such pre-expansion has been shown to activate oncogenes. This is caused by the exposure to an artificial environment and possible also to repetition of proliferation cycles that do not underlie normal control mechanisms of wound repair and remodeling. This artificial situation of course is not coherent with the body's capacity for regeneration following wounding and injury. Stem cell activation in man and stem cell commitment requires a full control of proliferation, while at the same time preventing oncogene activation.
These requirements are considered to be mandatory and their disregard in conventional teaching can cause the most severe and deleterious drawbacks of the current technology of in vitro cellular processes that inevitably are not only rather complicated but also risky for these reasons.
The other alternative represents a mere injection of stem cells, which is on the other hand no solution since the receiving site of the cells is highly variable and not fully controllable from the cell and scaffold transplanter's side. In all cases reported so far stem cells were at a closer look not fully achieving their original goal of resulting in an appropriate de novo tissue formation.
Accordingly there is need for controlling multipotency of stem cells after transplantation and at the time of transplantation. Past teaching was focusing on controls of multipotency rather before transplantation. A need also exists to avoid cell culture processes that may attempt to control cell differentiation but exhibit artificial side conditions that are harmful to the cells and are also not economical.
It is a great problem in carrying out these prior methods with respect to the quality and functionality of the transplant. Accordingly, there is demand for a method of abolishing all these limitations. The present invention was made in order to overcome the problems described, and to provide a practical method to rapidly engineer airway tissue and valves and in general all tissues of the animal or human body.